Loss of salivary gland function affects millions of people worldwide and is caused by various conditions including radiation therapy for head and neck cancers, surgical excision of salivary tumors, autoimmune diseases and cytotoxic insults. These patients have dramatically reduced salivary secretion, or hyposalivation, a condition that severely affects their quality of life. Currently, the only treatment option is pharmacologic management, but this is palliative and often less than satisfactory. As the secretory acinar cells are diminished by apoptosis or necrosis, restoration of salivary flow requires replacement of damaged or lost cells by either transplantation of stem/progenitor cells or by induction of endogenous stem/progenitor cells. At present, however, salivary gland stem cells have not been identified and we lack essential knowledge about the mechanism of cell renewal during normal homeostasis and injury-induced regeneration. These are central issues from the perspective of regenerative medicine targeted to salivary glands. In this regard, our laboratory was first to develop a method for in situ lineage tracing that we used to demonstrate the presence of stem cells in epidermis and sebaceous glands. We also showed that these cells functioned independently of hair follicle stem cells located in the bulge. We propose using a similar approach to identify the mechanism of cell maintenance and regeneration in salivary glands. Regeneration of secretory acinar cells in the salivary gland must occur via one of three mechanisms: self-duplication of functioning acinar cells in the gland, proliferation of ductal cell, as speculated, or derived from a population of immature, undifferentiated, slow-dividing stem cells. To explore the nature and location of glandular maintenance and renewal, we will use two novel genetic approaches: 1) we will generate bi- transgenic mice with regulated expression of H2B-GFP targeted to epithelial cells in salivary glands and perform long-term pulse-chase labeling to identify slow-dividing cells. These GFP-labeled cells will be characterized using antibodies selective for the major cell types of the salivary gland. We will then recover these slowly-cycling cells by flow cytometry and assess their regenerative abilities; 2) we will utilize lentiviral-mediated in situ genetic marking and fate mapping to elucidate the lineal relationship between various cells types including ductal and acinar cells during normal homeostasis and regeneration/repair in salivary glands. The identification and functional characterization of adult salivary gland stem cells and elucidation of the mechanism of gland renewal are critical steps toward developing effective strategies for regeneration of damaged salivary glands. The knowledge gained in this study will be a first step in a long-range plan to restore salivary secretory function using adult progenitor cells.